Advancement in wound management or tissue repair has been limited by the complexities, costs and limited efficacy of purified recombinant growth factors. Further, the combination of these recombinant proteins for more sophisticated approaches is complicated by the need for separate biologics. Optimally, we can move to a single biologic DNA pro-drug that provides for the generation of multiple growth factors that will work in concert with the required heparan sulfate co-receptor. Such a DNA pro-drug could be applied locally within a graft, bandage or gel to different tissue types including bone and skin, and the DNA pro- drug will have relatively greater stability than a comparable protein class of drugs making it ideal for field use. The platform DNA pro-drug could be modified easily will have relatively greater stability than a comparable protein class of drugs making it ideal for field use with greater affordability and wide-spread access. We will begin by testing pro-drugs expressing 2 or 3 growth factors designed for bone reconstruction and repair using a calcium phosphate graft scafold for sustained release. A significant aspect of this proposed technology is the co-expression of the required growth factor co-receptor for growth factor activity - a heparan sulfate proteoglycan. We have identified an elegant mechanism to provide this required growth factor co-receptor using a transgene that generates a site-specific heparan sulfate proteoglycan. DNA pro- drug delivery of this heparan sulfate transgene significantly enhanced growth factor activity and bone healing in the maxilla, providing a 3-fold dose enhancement of BMP-2 activity in preliminary experiments. Four US patents describing the essence of this technology have been awarded to Agenta Biotechnologies, Inc. The Specific Aims of this project are as follows: 1) create, characterize, and pre-clinically test efficacy of an expression plasmid that expresses both the heparan sulfate co-receptor and BMP-2;and 2) create, characterize, and pre-clinically test efficacy of an expression plasmid that expresses VEGF165 along with the heparan sulfate co-receptor and BMP-2, testing the additional need for rapid vascularization of a large avascular synthetic bone graft in a large, critical size graft area. In vivo, a well-characterized maxillary ridge augmentation model will deliver the experimental DNA pro-drugs on an approved calcium phosphate graft material to the surface of the bone with the goal of building new and unprecedented levels of additional bone that is fully integrated with the existing jaw. Primary in vitro outcomes include assessment of expression levels, and biologic function. Functional assays will measure osteoblast mineralization rates, cell proliferation, and endothelial cell vasculogenesis. Primary in vivo outcomes will be the volume and relative density of new bone or osteoid as measured using histomorphometric analysis. Secondary outcomes include histomorphometric assessments of inflammation, molecular evidence for in situ expression of the transgenes, and a pathology analysis. PUBLIC HEALTH RELEVANCE: Advancement of biologic therapeutics and the potential benefits they can provide in wound management and tissue regeneration has been limited by the complexities of manufacturing, regulation, stability, and partial efficacy of purified recombinant growth factors. The combination of these recombinant proteins for more sophisticated approaches to wound healing is complicated by the need for separate biologics. Optimally, we can move to a single biologic DNA pro-drug that provides for the generation of multiple growth factors together with the required heparan sulfate co-receptor for growth factors. In essence, this technology proposes multiple drug activities working in concert for tissue healing, regeneration, or remodeling, to be generated from a single pro-drug. Such a DNA pro-drug could be applied locally within a graft, bandage or gel for healing, regeneration, or remodeling of different tissue types including bone and skin, with engineered tissue-specific on-off controls for different growth factors. The platform DNA pro- drug could be modified easily and the DNA pro-drug will have relatively greater stability than a comparable protein class of drugs making it ideal for field use with greater affordability and wide-spread access. We will begin in this phase 1 project by testing pro-drugs expressing 2 or 3 biologics designed for bone reconstruction and repair. A significant aspect of this proposed technology is the co-expression of the required growth factor co-receptor for growth factor activity - a heparan sulfate proteoglycan. The proposed technology is a rational and timely approach to establish a new generation of therapeutics capable of cost-effectively and practically providing multiple biological drug activities for advanced wound management or tissue repair.